The physiological effects of EtOH appear to be due to its interaction with the cell membrane. Chronic exposure to EtOH induces changes in the membrane which lead to resistance to the effects of EtOH. However, controversy exists about the actual molecular changes occurring in the membrane which result in such tolerance to EtOH. We are studying neuronal cells in culture as a model system to determine the molecular mechanisms underlying the long-term changes after exposure to EtOH. We are using membrane enzymes which we can assay in intact, living cells to determine the time course for development of these changes. Since we use a homogeneous cell line in a defined medium where changes in EtOH concentration are the only variable, we will be able to observe changes in membrane function and lipid composition due to chronic treatment with EtOH. Moreover, since membrane lipid composition can be altered by changing the medium in which the cells grow, we will be able to selectively change membrane lipids and determine the role of individual lipids in the development of tolerance to EtOH. We will also attempt to prevent or reverse the development of tolerance by altering membrane lipid composition. During the period covered by this proposal, we will: characterize the acute and chronic effects of EtOH on adenosine-stimulated adenylate cyclase activity in these intact living cells; determine whether chronic exposure to EtOH also affects fatty acid, phospholipid or cholesterol composition of NG108-15 membranes; determine whether the changes in membrane lipid composition correlate with the observed changes in adenosine-stimulated adenylate cyclase activity; determine how withdrawal of EtOH affects both enzyme activities and lipid composition of NG108-15; determine whether changing the phospholipid and fatty acid composition of the membrane can reverse or prevent the changes produced by chronic treatment with EtOH.